1. Chapter 4 Molecular Cloning Methods

2. 4.1 Gene Cloning
Gene cloning is an indispensable molecular biology technique that allows scientists to produce large quantities of their gene of interest
Gene cloning links eukaryotic genes to small bacterial or phage DNAs and inserting these recombinant molecules into bacterial hosts
Gene cloning can produce large quantities of these genes in pure form

3. The Role of Restriction Endonucleases
Restriction endonucleases, first discovered in the late 1960s in E. coli, are named for preventing invasion by foreign DNA by cutting it into pieces
These enzymes cut at sites within the foreign DNA instead of chewing from the ends
By cutting DNA at specific sites they function as finely honed molecular knives

4. Naming Restriction Endonucleases
Restriction endonucleases are named using the 1st three letters of their name from the Latin name of their source microorganism Hind III
First letter is from the genus H from Haemophilus
Next two letters are the 1st two letters of the species name in from influenzae
Sometimes the strain designation is included “d” from strain Rd
If microorganism produces only 1 restriction enzyme, end the name with Roman numeral I Hind I
If more than one restriction enzyme is produced, the others are numbered sequentially II, III, IV, etc.

5. Restriction Endonuclease Specificity
Restriction endonucleases recognize a specific DNA sequence, cutting ONLY at that sequence
They recognize 4-bp, 6-bp, 8-bp palindromic sequences
The frequency of cuts lessens as the recognition sequence is longer
They cut DNA reproducibly in the same place

6. Restriction-Modification System
What prevents these enzymes from cutting up the host DNA?
They are paired with methylases
Theses enzymes recognize, methylate the same site
Together they are called a restriction-modification system, R-M system
Methylation protects DNA, after replication the parental strand is already methylated

7. An Experiment Using Restriction Endonuclease: Boyer and Cohen
An early experiment used EcoRI to cut 2 plasmids, small circular pieces of DNA independent of the host chromosome
Each plasmid had 1 EcoRI site
Cutting converted circular plasmids into linear DNA with the same sticky ends
The ends base pair
Some ends re-close
Others join the 2 pieces
DNA ligase joins 2 pieces with covalent bonds

8. Vectors
Vectors function as DNA carriers to allow replication of recombinant DNAs
Typical experiment uses 1 vector plus a piece of foreign DNA
The inserted and foreign DNA depends on the vector for its replication as it does not have an origin of replication, the site where DNA replication begins
There are 2 major classes of vectors:
Plasmids
Phages

9. Plasmids As Vectors
pBR plasmids were developed early but are rarely used today
pUC series is similar to pBR
40% of the DNA has been deleted
Cloning sites are clustered together into one area called the multiple cloning site (MCS)
MCS allows one to cut the vector and foreign gene with two different restriction enzymes and use a directional cloning technique to know the orientation of the insert

12. Screening: antibiotics and b-galactosidase
Screening capabilities within plasmids:
Antibiotic resistance genes (i.e., ampicillin resistance gene) allow for the selection of bacteria that have received a copy of the vector
Multiple cloning site inserted into the gene lacZ’ coding for the enzyme b-galactosidase
Clones with foreign DNA in the MCS disrupt the ability of the cells to make b-galactosidase
Plate on media with a b-galactosidase indicator (X-gal) and clones with intact b-galactosidase enzyme will produce blue colonies
Colorless colonies should contain the plasmid with foreign DNA compared to blue colonies that do not contain the plasmid with DNA

13. Phages As Vectors
Bacteriophages are natural vectors that transduce bacterial DNA from one cell to another
Phage vectors infect cells much more efficiently than plasmids transform cells
Clones are not colonies of cells using phage vectors, but rather plaques, a clearing of the bacterial lawn due to phage killing the bacteria in that area

15. l Phage Vectors
First phage vectors were constructed by Fred Blattner and colleagues
Modifications included removal of the middle region and retention of the genes needed for phage replication
Could replace removed phage genes with foreign DNA
Advantage: Phage vectors can receive larger amounts of foreign DNA (up to 20kb of DNA)
Traditional plasmid vectors take much less
Phage vectors require a minimum size foreign DNA piece (12 kb) inserted to package into a phage particle

16. Cloning Using a Phage Vector

17. Genomic Libraries
A genomic library contains clones of all the genes from a species genome
Restriction fragments of a genome can be packaged into phage using about 16 – 20 kb per fragment
This fragment size will include the entirety of most eukaryotic genes
Once a library is established, it can be used to search for any gene of interest

18. Selection via Plaque Hybridization
Searching a genomic library requires a probe to determine which clone contains the desired gene
Ideal probe – labeled nucleic acid with sequence matching the gene of interest

19. Cosmids Cosmids are designed for cloning large DNA fragments
Behave both as plasmid and phage and contain
cos sites, cohesive ends of phage DNA that allow the DNA to be packaged into a l phage head
Plasmid origin of replication permitting replication as plasmid in bacteria
Nearly all l genome removed so there is room for large inserts (40-50 kb)
Very little phage DNA yields them unable to replicate, but they are infectious and carry their recombinant DNA into bacterial cells

20. M13 Phage Vectors Long, thin, filamentous phage Contains: Advantage
Gene fragment with b-galactosidase
Multiple cloning site like the pUC family
Advantage
This phage’s genome is single-stranded DNA
Fragments cloned into it will be recovered in single-stranded form

21. M13 Cloning to Recover Single-stranded DNA Product
After infecting E. coli cells, single-stranded phage DNA is converted to double-stranded replicative form (RF)
Use the replicative form for cloning foreign DNA into MCS
Recombinant DNA infects host cells resulting in single-stranded recombinant DNA
Phage particles, containing single-stranded phage DNA is secreted from transformed cells and can be collected from media

22. Phagemids Phagemids are also vectors
Like cosmids have aspects of both phages and plasmids
Has MCS inserted into lacZ’ gene to screen blue/ white colonies
Has origin of replication of single-stranded phage f1 to permit recovery of single-stranded recombinant DNA
MCS has 2 phage RNA polymerase promoters, 1 on each side of MCS

23. Eukaryotic Vectors and Very High Capacity Vectors
There are vectors designed for cloning genes into eukaryotic cells
Other vectors are based on the Ti plasmid to carry genes into plant cells
Yeast artificial chromosomes (YAC) and bacterial artificial chromosomes (BAC) are used for cloning huge pieces of DNA

24. Identifying a Specific Clone With a Specific Probe
Probes are used to identify a desired clone from among the thousands of irrelevant ones
Two types are widely used
Polynucleotides (also called oligonucleotides)
Antibodies

25. Polynucleotide Probes
Looking for the gene you want, you might use the homologous gene from another organism
If already cloned and there is enough sequence similarity to permit hybridization
Need to lower stringency of hybridization conditions to tolerate some mismatches
High temperature, high organic solvent concentration and low salt concentration are factors that promote separation of two strands in a DNA double helix and can be adjusted as needed

26. Protein-based Polynucleotide Probes
No homologous DNA from another organism?
If amino acid sequence is known, deduce a set of nucleotide sequences to code for these amino acids
Construct these nucleotide sequences chemically using the synthetic probes
Why use several?
Genetic code is degenerate with most amino acids having more than 1 nucleic acid triplet
Must construct several different nucleotide sequences for most amino acids

27. cDNA Cloning
cDNA - complementary DNA or copy DNA that is a DNA copy of RNA
A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time
Such a library can contain tens of thousands of different clones

28. Making a cDNA Library

29. Reverse Transcriptase
Central to successful cloning is the synthesis of cDNA from an mRNA template using reverse transcriptase (RT), an RNA-dependent DNA polymerase
RT cannot initiate DNA synthesis without a primer
Use the poly(A) tail at 3’ end of most eukaryotic mRNA so that oligo(dT) may serve as primer

30. Ribonuclease H
RT with oligo(dT) primer has made a single-stranded DNA off of mRNA
Need to remove the RNA
Partially degrade the mRNA using ribonuclease H (RNase H)
Enzyme degrades RNA strand of an RNA-DNA hybrid
Remaining RNA fragments serve as primers for “second strand” DNA using nick translation

31. Nick Translation The nick translation process simultaneously:
Removes DNA ahead of a nick
Synthesizes DNA behind nick
Net result moves the nick in the 5’ to 3’ direction
Enzyme often used is E. coli DNA polymerase I
Has 5’ to 3’ exonuclease activity
Allows enzyme to degrade DNA ahead of the nick

32. Terminal Transferase
cDNAs don’t have the sticky ends of genomic DNA cleaved with restriction enzymes
Blunt ends will ligate, but is inefficient
Generate sticky ends using enzyme terminal deoxynucleotidyl transferase (TdT), terminal transferase with one dNTP
If use dCTP with the enzyme
dCMPs are added one at a time to 3’ ends of the cDNA
Same technique adds oligo(dG) ends to vector
Generate ligation product ready for transformation

33. Rapid Amplification of cDNA Ends
If generated cDNA is not full-length, missing pieces can be filled in using rapid amplification of cDNA ends (RACE)
Technique can be used to fill in either the missing portion at the 5’-end (usual problem)
Analogous technique can be used to fill in a missing 3’-end

34. RACE Procedure
Use RNA prep containing mRNA of interest and the partial cDNA
Anneal mRNA with the incomplete cDNA
Reverse transcriptase will copy rest of the mRNA
Tail the completed cDNA with terminal transferase using oligo(dC)
Second strand synthesis primed with oligo(dG)

35. 4.2 The Polymerase Chain Reaction
Polymerase chain reaction (PCR) is used to amplify DNA and can be used to yield a DNA fragment for cloning
Invented by Kary Mullis and colleagues in 1980s
Special heat-stable polymerases are now used that are able to work after high temperatures - researchers no longer need to add fresh DNA polymerase after each round of replication

36. Standard PCR
Use enzyme DNA polymerase to copy a selected region of DNA
Add short pieces of DNA (primers) that hybridize to DNA sequences on either side of piece of interest – causes initiation of DNA synthesis through that area, X
Copies of both strands of X and original DNA strands are templates for the next round of DNA synthesis
The selected region of DNA now doubles in amount with each synthesis cycle

37. Amplifying DNA by PCR

38. Using Reverse Transcriptase PCR (RT-PCR) in cDNA Cloning
To clone a cDNA from just one mRNA whose sequence is known, a type of PCR called reverse transcriptase PCR (RT-PCR) can be used
Difference between PCR and RT-PCR
Starts with an mRNA, not dsDNA
Begin by converting mRNA to DNA
Use forward primers to convert ssDNA to dsDNA
Continue with standard PCR

39. RT-PCR to clone a single cDNA
With care, restriction enzyme sites can even be added to the ends of the cDNA of interest
Able to generate sticky ends for ligation into vector of choice
2 sticky ends permits directional cloning

40. Real-Time PCR
Real-time PCR quantifies the amplification of the DNA as it occurs
As the DNA strands separate, they anneal to forward and reverse primers, and to a fluorescent-tagged oligonucleotide complementary to part of one DNA strand that serves as a reporter probe

41. Real-Time PCR
A fluorescent-tagged oligonucleotide serves as a reporter probe
Fluorescent tag at 5’-end
Fluorescence quenching tag at 3’-end
As PCR progresses from the forward primer the 5’ tag is separated from the 3’ tag and allows the 5’ tag to fluoresce
Fluorescence increases with incorporation into DNA product and can be quantitated

42. 4.3 Methods of Expressing Cloned Genes
Cloning a gene permits
Production of large quantities of a particular DNA sequence for detailed study
Large quantities of the gene’s product can also be obtained for further use
Study
Commerce

43. Expression Vectors
Vectors discussed so far are used to first put a foreign DNA into a bacterium to replicate and screen
Expression vectors are those that can yield protein products of the cloned genes
Bacterial expression vectors typically have two elements required for active gene expression; a strong promoter and a ribosome binding site near an initiating codon

44. Fusion Proteins
Some cloning vectors, pUC and pBS, can work as expression vectors using lac promoter
If inserted DNA is in the same reading frame as interrupted gene, a fusion protein results
These have a partial b-galactosidase sequence at amino end
Inserted cDNA protein sequence at carboxyl end

45. Inducible Expression Vectors
Main function of expression vector is to yield the product of a gene – usually more is better
For this reason, expression vectors have very strong promoters
It is usually advantageous to keep a cloned gene repressed until time to express
Large quantities of eukaryotic protein in bacteria are usually toxic
Can accumulate to levels that interfere with bacterial growth
Expressed protein may form insoluble aggregates, called inclusion bodies

46. Controlling the lac Promoter
lac promoter is somewhat inducible
Stays off until stimulated by inducer IPTG
However, repression is typically incomplete or leaky and some expression will still occur
To avoid this problem, use a plasmid or phagemid carrying its own lacI repressor gene to keep the cloned gene off until it is induced by IPTG

47. Alternatives to the lac Promoter
The hybrid trc promoter combines the strength of the trp (tryptophan operon) promoter with the inducibility of the lac promoter
Promoter from ara operon, PBAD, allow fine control of transcription
Inducible by arabinose, a sugar
Transcription rate varies with arabinose concentration

48. Alternatives to the lac Promoter
The lambda (l) phage promoter, PL, is tightly controlled
Expression vectors with this promoter-operator system are used in host cells with temperature-sensitive l repressor gene
Repressor functions at low temperatures
Raise temperature above the nonpermissive level (42’C) and the repressor doesn’t function and the cloned gene is expressed

49. Expression Vectors That Produce Fusion Proteins
Most vectors express fusion proteins
The actual natural product of the gene isn’t made
Extra amino acids help in purifying the protein product
Oligohistidine expression vector has a short sequence just upstream of MCS encoding 6 His
Oligohistidine has a high affinity for divalent metal ions like nickel (Ni2+)
Permits purification by nickel affinity chromatography
The his tag can be removed using enzyme enterokinase without damage to the protein product

50. Using an Oligohistidine Expression Vector

51. Expression vector lgt11
This phage contains the lac control region followed by the lacZ gene
The cloning sites are located within the lacZ gene
Products of gene correctly inserted will be fusion proteins with a b-galactosidase leader

52. Detecting positive lgt11 clones via antibody screening
Lambda phages with cDNA inserts are plated
Protein released are blotted onto a support
Probe with antibody specific to protein
Antibody bound to protein from plaque is detected with labeled protein A

53. Bacterial Expression System Shortcomings
There are problems with expression of eukaryotic proteins in a bacterial system
Bacteria may recognize the proteins as foreign and destroy them
Post-translational modifications are different in bacteria
Bacterial environment may not permit correct protein folding
Very high levels of cloned eukaryotic proteins can be expressed in useless, insoluble form

54. Eukaryotic Expression Systems
Avoid bacterial expression problems by expressing the protein in a eukaryotic cell
Initial cloning done in E. coli using a shuttle vector, able to replicate in both bacterial and eukaryotic cells
Yeast is suited for this purpose
Rapid growth and ease of culture
A eukaryote with more appropriate post-translational modification
Use of the yeast export signal peptide secretes protein into growth medium for easy purification

55. Use of Baculovirus As Expression Vector
Viruses in this class have a large circular DNA genome, 130 kb
Major viral structural protein is made in huge amounts in infected cells
The promoter for this protein, polyhedrin, is very active
These vectors can produce up to 0.5 g of protein per liter of medium
Nonrecombinant viral DNA entering cells does not result in infectious virus as it lacks an essential gene supplied by the vector

56. Expressing a Gene in a Baculovirus

57. Animal Cell Transfection
Carried out in two ways:
Calcium phosphate
Mix cells with DNA in a phosphate buffer and add a solution of calcium salt to form a precipitate
The cells take up the calcium phosphate crystals, which include some DNA
Liposomes
The DNA is mixed with lipid to form liposomes, small vesicles with some of the DNA inside
DNA-bearing liposomes fuse with the cell membrane to deliver DNA inside the cell

58. Using the Ti Plasmid to Transfer Genes to Plants
Genes can be introduced into plants with vectors that can replicate in plant cells
Common bacterial vector promoters and replication origins are not recognized by plant cells
Plasmids are used containing T-DNA
T-DNA is derived from a plasmid known as tumor-inducing (Ti)
Ti plasmid comes from bacteria that cause plant tumors called crown galls

59. Ti Plasmid Infection
Bacterium infects plant, transfers Ti plasmid to host cells
T-DNA integrates into the plant DNA causing abnormal proliferation of plant cells
T-DNA genes direct the synthesis of unusual organic acids, opines which can serve as an energy source to the infecting bacteria but are useless to the plant

60. The Ti Plasmid Transfers Crown Gall

61. Use of the T-DNA Plasmid